Despite studies based on experimental models and clinical trials, the impact of T cell avidity on tumor immunity remains incompletely understood. It is generally believed that higher avidity T cells would be more successful for cancer immunotherapy, but concern remains about loss of specificity, self-reactivity, and increased susceptibility to immune suppression or apoptosis. We previously reported that sensitization of mice to a melanoma differentiation antigen, tyrosinase-related protein-2 (TRP-2, dopachrome tautomerase, Dct), resulted in weak T cell responses unless combined with GM-CSF administration and CTLA-4 blockade. In collaboration with Enzo Bronte (Univ. Padua), we subsequently established three T cell lines with specificity for the TRP-2(180-188) peptide epitope, yet differed in their 'avidity', which was defined by their ability to respond to varied doses of antigen. We cloned the T cell antigen receptor (TcR) genes from these cell lines and used them to make transgenic (Tg) mice. This generated a unique model system as it consists of distinct TcR gene combinations that confer common antigenic specificity. The goal of this project is to use these novel transgenic mouse models to test the hypothesis that T cell avidity is a critical determinant of tumor immunity and regulates susceptibility to immune suppression. These studies will identify how T cell avidity correlates with tumor immunity and autoimmunity and lead to more effective cancer therapies. We previously reported that the intermediate avidity Tg mouse line did not spontaneously develop autoimmune depigmentation, despite systemic expression of TRP-2 in the skin. Peripheral T cells from these TCR Tg mice exhibited a naive phenotype and proliferated in response to TRP-2 in vitro. However, transfer of in vitro-activated Tg T cells reduced B16 pulmonary tumor burden. Adoptive transfer of naive TcR Tg T cells into wild-type C57BL/6 mice, in combination with a TRP-2-pulsed dendritic cell (DC) vaccine, induced proliferation of the Tg T cells and resulted in migration of the Tg T cells into subcutaneous B16 melanoma tumors. Although these tumor-infiltrating Tg T cells remained reactive against TRP-2, they did not reduce growth of the primary tumor. These data demonstrate that despite in vivo priming and retention of effector function, tumor-infiltrating T cells may fail to reduce tumor burden. This lack of response to tumor-derived antigen, and the subsequent lack of tumor reduction suggest that the T cells are ignorant of the developing tumor. In 2013, we reported on the higher avidity Tg T cells. Mice bearing these T cells spontaneously develop autoimmune depigmentation, suggesting that the Tg T cells are primed by the endogenous TRP-2 antigen, resulting in T cell-mediated killing of the TRP-2-expressing melanocytes. In addition, B16 melanoma tumor growth is reduced in the TcR Tg mice. When the high avidity Tg T cells are transferred into wild-type mice and primed with a peptide-pulsed DC vaccine, B16 melanoma growth is significantly slowed. Interestingly, unlike the intermediate avidity T cells, the high avidity T cells become tolerized within the tumor microenvironment, as they lose their ability to secrete interferon-gamma and display lytic potential. In parallel, there is a loss of MHC expression on the tumor cells, suggesting that in the absence of interferon-gamma production by Tg T cells, the tumor cells lose their susceptibility to killing and evade immune recognition. In addition, we have noted that provision of CD4+ T cells preferentially helps the higher avidity T cells, improving immunity to recently established tumors, at the cost of increased autoimmune depigmentation. On-going studies will identify the different gene expression patterns of these two populations of cells. Identifying patterns that associate with susceptibility to tolerization may provide novel targets for maintaining more durable T cell responses. Finally, because the generation of effective T cell memory was shown to be a function of metabolic status, we are characterizing the metabolic profiles of these two divergent populations of T cells and we will correlate the profile with effector function and susceptibility to tolerization. This will enable us to identify additional differences between the two populations. More recently, we have begun to study the lowest avidity Tg T cells. Of greatest interest is our observation that these cells arise in the Tg mice with a phenotype consistent with antigen-experienced or memory T cells. In preliminary studies, these cells also seem capable of reducing B16 tumor growth and secrete cytokines in the absence of TcR ligation. These observations are consistent with a newly emerging population of memory T cells referred to endogenous, or virtual, memory T cells. Thus, our on-going studies are examining how these endogenous memory cells differ from traditional memory cells, determining how they control tumor growth (given their low avidity), and whether they are also susceptible to tolerization in the tumor microenvironment.